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European Journal of Medicinal Chemistry 48 (2012) 231e243

Contents lists available

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Cytotoxic potential of novel 6,7-dimethoxyquinazolines

Mange R. Yadav a,*, Fedora Grande b,c, Bishram S. Chouhan a, Prashant P. Naik a, Rajani Giridhar a,Antonio Garofalo c, Nouri Neamati b

a Pharmacy Department, The M.S. University of Baroda, Vadodara, 390001 Gujarat, IndiabDepartment of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089, USAcDipartimento di Scienze Farmaceutiche, Universitadella Calabria, 87036 Arcavacata di Rende (Cs), Italy

a r t i c l e i n f o

Article history:Received 17 January 2011Received in revised form23 July 2011Accepted 10 December 2011Available online 21 December 2011

Keywords:QuinazolinesQuinazolinonesColon cancerAnticancer agentsOvarian cancer

* Corresponding author. Tel.: þ91 265 2434187; faxE-mail address: mryadav11@yahoo.co.in (M.R. Yad

0223-5234/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.ejmech.2011.12.020

a b s t r a c t

Herein, we report the synthesis and cytotoxicity of a series of substituted 6,7-dimethoxyquinazolinederivatives. The cytotoxic activity of all synthesized compounds has been evaluated againstHCT116p53þ/þ and HCT116p53�/� colon cancer cells and a HEY ovarian cancer cell line naturally resistantto cisplatin. Nine of the tested compounds showed significant cytotoxicity in all cell lines at 10 mM. Themost promising derivative (7c) showed IC50values of 0.7 and 1.7 mM in the two colon cancer cell lines.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

2-Phenyl-4-quinolones (PQ) [1e4] and several flavonoids (FL)[5] are known to possess potent antimitotic activity (Fig. 1). Thesetwo classes of compounds, sharing a similar overall skeleton anddiffering in the nature of the heteroatoms, seem to act with thesame mechanism based on kinase inhibition or the prevention oftubulin polymerization. The effects observed were similar to thoserecorded for antimitotic natural products such as colchicine,podophyllotoxin, and combretastatin 4-A. Thus due to their simplestructural features, these compounds represent interesting candi-dates to be developed as antitumor agents. Lee and co-workersreported the synthesis and biological activity of variouslysubstituted 2-arylquinazolinones (AQ) [6], which were consideredas bioisosteres of PQ and FL. AQ were found to possess significantgrowth inhibitory action against a panel of colon cancer cell lines[7e9]. It was established that the contemporary presence of anaromatic substituent on position-2 of the quinazoline nucleus and6,7-dimethoxy substitution are necessary for activity [7,10].Amongst all the tested derivatives, compound AQ1 showed thehighest cytotoxic activity with an ED50¼1.6 mM against a humanepidermoid carcinoma cell line KB-VIN [6]. This inspired us to use

: þ91 265 2418927.av).

son SAS. All rights reserved.

compound AQ1 as a lead molecule for the development of morepotent anticancer agents with desirable pharmacokineticproperties.

In continuation of our studies aimed at the identification ofsmall molecules with cytotoxic properties, we planned thesynthesis of several quinazolin-4-one derivatives, maintaining the6,7-dimethoxy substituents. In particular, 2-aryl, -benzyl, -aryla-mino and arylaminomethyl derivatives were prepared. Replace-ment of the oxygen carbonyl moiety of quinazolinone system by anamino or azido groupwas also explored. Several derivatives bearinga lipophilic n-butyl group at position-3 of the quinazoline systemwere also prepared.

2. Results and discussion

2.1. Chemical

Firstly 2-aryl-6,7-dimethoxy-4(3H)-quinazolinones (4) havebeen synthesized as reported in Scheme1. Substituted benzoic acids(1) were condensed with 2-amino-4,5-dimethoxy benzamide (2) inpresence of EDC or thionyl chloride to obtain the diamides (3). Thediamides were subjected to cyclization under different experi-mental conditions to afford the desired 2-aryl-6,7-dimethoxyquinazolin-4(3H)-ones (4).

A similar strategy was utilized for the synthesis of compounds(7), inwhich the aryl group was linked to the quinazolinone system

COOH

NH2

NH2

OMeO

MeO NH

NH2

OMeO

MeO

OX

XN

NH

OMeO

MeO X

+

X = a) 3-NHCOMe; b) 2-Cl; c) 3-Cl; d) 3-OMe; e) 4-OMe; f) 3-Me; g) 4-Me

i ii

(1 ) (2) (3) (4)

Scheme 1. Reagents and conditions: i) EDC, TEA, CHCl3 or SOCl2, 1,4 Dioxane; ii) Fuse or NaOH, CH3OH, or t-BuONa, ethylene glycol, MW.

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243232

by a methylene group. Substituted phenylacetic acids (5) werecondensed with the amide (2) to obtain the diamides (6), whichgave the desired quinazolines (7) under basic conditions andmicrowave irradiation (Scheme 2).

A second series of compounds in which the quinazolinone andthe aryl moieties were linked by aminomethylene moiety wereobtained as depicted in Scheme 3. The methyl anthranilate deriv-ative (8) was reacted with chloroacetonitrile in presence of dryhydrogen chloride to afford the 2-chloromethyl derivative (9),which gave the desired products (11) after reaction with theappropriate substituted anilines (10).

An n-butyl group was then introduced into position-3 of thequinazolinone moiety in order to improve lipophilicity ofcompounds (4) to give derivatives (16), according to Scheme 4. Thebenzoic acid derivatives (12) were transformed to acid chlorides bytreatment with thionyl chloride. The acid chlorides were thentreated with n-butylamine to give the desired amides (13). Thenitro group in compounds (13) was reduced with iron powder togive the desired amines (14). The acid chlorides obtained from thebenzoic acid derivatives (1) were reacted with the amino deriva-tives (14) to provide the diamides (15), which were cyclized underbasic conditions to the desired products (16) (Scheme 4). Thenitriles were converted into the tetrazoles (15f and 15g from 15dand 15e, respectively), which were cyclized to the desired products(16f and 16g).

It was also planned to replace the oxygen of the 4-keto group ofquinazolinone ring with a basic amino group. Accordingly, the 4-keto derivatives (4) were treated with phosphorous oxychlorideto give the corresponding 4-chloro derivatives (17), which in turnwere converted into the azide derivatives (18) by treatment withsodium azide. Finally, the azide was reduced into correspondingamines (19) by catalytic hydrogenation (Scheme 5).

NH

O

R

R1

PQ

O

O

R

R1

FL

N

NH

O

R

R1

AQ

N

NH

OMeO

MeO

AQ1

Fig. 1. General structures of reference compounds 2-phenyl-4-quinolones PQ, flavo-noids FL, 2-arylquinazolinones AQ, and the structure of the lead compound AQ1.

Replacement of the methylene bridge with a basic aminofunction was also taken into consideration. The 4-amino-2-chloro-quinazoline (21) [11] was reacted with substituted anilines (20) togive compounds (22), as reported in Scheme 6.

Physical data of the final synthesized final compounds issummarized in Table 1.

2.2. Cytotoxicity

Twenty-nine compounds selected on the basis of their struc-tural features were initially tested in three human cancer cell linesfrom different tumor origins, two colon cancer cells HCT116p53þ/þ

and HCT116p53�/�, and an ovarian cancer cell line HEY, naturallyresistant to cisplatin. Nine compounds (7ae7c, 18b, 18c, 18ee18gand 22b) showed >50% cytotoxicity at 10 mM (Table 2) in one ormore of the cell lines. These compounds were tested at multipledoses to determine their corresponding IC50 values (Table 3).

Compound 7c was found to be the most potent derivativeagainst HCT116p53þ/þ and HCT116p53�/� cell lines with IC50 valuesof 1.7 and 0.7 mM, respectively.

Compound 22b was the second most potent derivative againstthe same cell lines with IC50 values of 6 and 5 mM, respectively.Compounds 18b, 18c, 18e, 18f, 18g, 7a and 7b showed onlymoderate activities against HCT116 p53þ/þ cells while compound18c, 18e and 18g showed moderate activity against HCT116 p53�/�

cells.Considering their cytotoxicity profile, we have examined the

effect of compounds 7c and 22b on tumor cell viability, usingcolony formation assay. The exposure of the two colon cancer celllines to micromolar concentrations of these compounds resulted ina significant inhibition of colony formation. Compounds 7c and 22bcompletely abolished cell growth, at doses above 1 and 5 mM,respectively (Fig. 2).

Cell cycle perturbation induced by compound 22b was exam-ined in HCT116 p53þ/þ and HCT116 p53�/� colon cancer cells. Theanalysis of DNA profile by flow cytometry indicated that compound22b induced partial cell cycle arrest in G2/M phase in colon cancercell lines. Treatment with compound 22b caused retention of 34.5%of cells in G2/M phase in the HCT116 p53�/� cell, after 48 h treat-ment compared to 24.5 in the controls. The property of thesecompounds to induce cell cycle arrest makes them promisingagents for combination with drugs acting at different stages of cellcycle (Fig. 3).

A few considerations can be drawn regarding thestructureeactivity relationships for the newly synthesized class ofcompounds. It seems that the presence of the oxygen or the aminogroup at position 4 does not significantly impair the cell growthinhibitory activity. Compounds bearing a one-atom linker (7ae7c,22b) between the quinazolinone system and the aryl ring showedhigher activity. The n-butyl lipophilic moiety, at position-3 of thequinazoline ring, adversely affects the cell inhibiting activity. The

NH2

NH2

OMeO

MeO NH

NH2

OMeO

MeO

OAr

N

NH

OMeO

MeOAr

Cl Cl

+i ii

(5) (2) (6) (7)

ArCH2COOH

Ar = a) ; b) ; c)

Scheme 2. Reagents and conditions: i) SOCl2, 1,4 Dioxane; ii) t-BuONa, ethylene glycol, MW.

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243 233

best structural combination required for activity seems to be thepresence of an aryl group, substituted or not, appended to thequinazoline system by a methylene or an NH group.

3. Conclusion

In this study, we designed and synthesized a series ofsubstituted 6,7-dimethoxyquinazoline derivatives. The newlysynthesized compounds have been tested against three humancancer cell lines for initial cytotoxicity evaluation.Structureeactivity relationship study revealed that the substitutionof the hydrogen at position-3 with a lipophilic n-butyl groupreduces the activity, whereas the introduction of a bulky aromaticgroup as substituent at position-2 of the quinazoline ring, isfavorable for cell inhibitory activity. In particular, the twocompounds 7c and 22b showed an interesting cytotoxic profile.Their biological activities were further assessed by colony forma-tion assay to confirm cytotoxicity. Flow cytometry analysisdemonstrated that compound 22b exerted cell cycle arrest inHCT116 colon cancer cells. Considering their cytotoxicity profiles,these compounds seem to have potentials as novel

NH2

OMeO

MeO

OMe MeO

MeO

N

OMeO

MeO

X = a) 4-COOH; b) 3-NHSO2M

i

(8)

(11)

Scheme 3. Reagents and conditions: i) ClCH2CN

chemotherapeutics and, therefore, warrant further preclinical andmechanistic investigation.

4. Experimental

4.1. Chemistry

Melting points were determined using a Veego make silicon oilbath-type melting point apparatus and are uncorrected. Purity ofthe compounds and completion of reactions were monitored bythin layer chromatography (TLC) on silica gel plates (60 F254;Merck), visualizing with ultraviolet light or iodine vapors. The IRspectra were recorded using KBr disc method on a Shimadzu FT-IR,Model 8300. The 1H NMR spectra (on a Bruker 300/400 MHzspectrometer) were recorded in DMSO. Chemical shifts were re-ported as part per million (d ppm) using tetramethylsilane (TMS) asan internal standard. The assignment of exchangeable protons wasconfirmed by the D2O exchange studies wherever required. Massspectral data was obtained on a QTRAP Applied Biosystem SCIEXspectrometer or on JEOL-SX-102 instrument (JEOL, Tokyo, Japan) byfast atom bombardment (FAB positive mode). Elemental analyseswere obtained on Carlo Erba, Italy and PerkineElmer instruments.

N

NH

O

Cl

NHNH

NH2

X

X

e; c) 4-NHSO2Me; d) 4-OMe

(9)

ii

(10)

+

, Dry HCl gas, 1,4 Dioxane; ii) K2CO3, DMF.

N

NH

OMeO

MeO

O X

OH

OMeO

MeO NO2

NH

OMeO

MeO NO2

NH

OMeO

MeO NH2

N

N

OMeO

MeOX

-n.Bu

-n.Bu-n.Bu

-n.Buiii

(16)

X = a) 4-NHSO2Me; b) 3-NHSO2Me; c) 4-NHCOMe; d) 4-CN; e) 3-CN; f) 4-(1H-tetrazol-5-yl); g) 3-(1H-tetrazol-5-yl)

iii

iv

(12) (13) (14)

(1)

+

(15)

Scheme 4. Reagents and conditions: i) SOCl2, n-BuNH2, THF; ii) Fe, NH4Cl, CH3OH; iii) SOCl2, TEA, 1,4 Dioxane; iv) NaOH, CH3OH.

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243234

Spectral data (IR, NMR, and MS) confirmed the structures of thesynthesized compounds and the purity were ascertained bymicroanalysis. Microwave reactions were performed in CEM-Discovery, USA microwave reactor.

4.1.1. 2-(3-Acetamidobenzamido)-4,5-dimethoxybenzamide (3a)3-Acetamidobenzoic acid (1a) (0.46 g, 2.55 mmol) was dissolved

in chloroform (50 mL) and the solution was cooled to 10e15 �C. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC) (0.59 g, 3.06 mmol) was added in portions and the reactionmixture was stirred for 45 min at 10e15 �C. 2-Amino-4,5-dimethoxybenzamide (2) (0.5 g, 2.55 mmol) and triethylamine(TEA) (0.88 mL, 6.38 mmol) were subsequently added at 10e15 �C.The reaction mixture was stirred at rt for 15 h, washed witha solution of 5% hydrochloric acid (2� 50 mL), aq sodium

N

N

ClMeO

MeOX

N

NMeO

MeO

N3

X N

N

NH2MeO

MeO X

N

NH

OMeO

MeO X

iii

(17)

(18) (19)

(4)

i

ii

X = b) 2-Cl; c) 3-Cl; d) 3-OMe; e) 4-OMe; f) 3-Me; g) 4-Me

Scheme 5. Reagents and conditions: i) POCl3, DMF; ii) NaN3, DMF, mw; iii) H2/Pd/(C),CH3OH.

bicarbonate (5%, 2� 50 mL) and water (2� 50 mL), sequentially.The organic layer was dried, concentrated under reduced pressureand the crude solid so obtained was purified by recrystallizationfrom chloroformemethanol to afford compound 3a (0.65 g, 72%):mp 195e197 �C (dec.); IR (KBr): 3271, 1668, 1633, 1539, 1404, 1313,1242, 1180 and 1008 cm�1.

4.1.2. 2-(2-Chlorobenzamido)-4,5-dimethoxybenzamide (3b)A mixture of 2-chlorobenzoic acid (1b) (0.61 g, 3.92 mmol) and

thionyl chloride (1 mL) was heated for 2 h. The excess of thionylchloride was recovered under reduced pressure and the residue soobtainedwas dissolved in dry dioxane (1 mL). The solutionwas thendropwise added to mixture of 2-amino-4,5-dimethoxybenzamide(2) (0.7 g, 3.57 mmol) and TEA (1.2 mL, 8.92 mmol) in dry dioxane(2 mL) at 10 �C. The reactionmixturewas stirred at rt for another 1 hand quenched into cold water (50 mL). The precipitate formed wasfiltered, washed with water and dried to obtain compound 3b(0.85 g, 71%): mp 213e215 �C (dec.); IR (KBr,): 3406, 3305, 1658,1645, 1612, 1525, 1253, 1078 and 1039 cm�1.

4.1.3. 2-(3-Chlorobenzamido)-4,5-dimethoxybenzamide (3c)Following a procedure identical to the one as described for 3b,

but using 3-chlorobenzoic acid (1c) (0.60 g, 3.92 mmol), compound3c was obtained as a pale yellow solid (0.60 g, 50%): mp

N

N

NH2

MeO

MeO NH

NH2

XN

NMeO

MeO Cl

NH2

X

NHSO2Me

+DMF

X = a) 4-NO2; b)

(20)(21) (22)

Scheme 6.

Table 1Chemical structure and physicochemical data of final compounds.

Structures No. X Mol. wt. Mol. formula Mp (�C) IR (KBr)cm�1

N

NH

OMeO

MeO X(4)

4a 3-NHCOMe 339.35 C18H17N3O4 >310 3301, 1658, 1625, 1604, 1533, 1436, 1236, 1168and 1010

4b 2-Cl 316.75 C16H13ClN2O3 275e277 (dec.) 1654, 1614, 1488, 1438, 1388, 1282, 1215, 1176and 1028

4c 3-Cl 316.75 C16H13ClN2O3 >310 1652, 1614, 1596, 1490, 1390, 1276, 1178, 1103and 1031

4d 3-OMe 312.33 C17H16N2O4 262e264 (dec.) [12] 1651, 1616, 1494, 1436, 1390, 1284, 1249, 1053and 1029

4e 4-OMe 312.33 C17H16N2O4 255e258 (dec.) 1665, 1619, 1490, 1441, 1386, 1304, 1252, 1176and 1030

4f 3-Me 296.33 C17H16N2O3 278e280 (dec.) 1652, 1610, 1492, 1434, 1388, 1278, 1178 and1037

4g 4-Me 296.33 C17H16N2O3 285e287 (dec.) 1662, 1610, 1496, 1436, 1388, 1244, 1174 and1033

N

NH

OMeO

MeOAr

(7)

7a

Cl330.77 C17H15ClN2O3 245e249 (dec.) 1668, 1612, 1488, 1286, 1236, 1218, 1099, 1166

and 1037

7b

Cl330.77 C17H15ClN2O3 243e245 (dec.) 1665, 1612, 1558, 1510, 1473, 1390, 1242, 1176

and 1034

7c 346.39 C21H18N2O3 262e264 (dec.) 1666, 1612, 1434, 1390, 1274, 1242, 1218, 1078and 1037

N

NH

OMeO

MeONH

X(11)

11a 4-COOH 355.35 C18H17N3O5 229e231 (dec.) 3373, 1677, 1648, 1604, 1487, 1386, 1271, 1172and 1010

11b 3-NHSO2Me 404.45 C18H20N4O5S 297e299 3222, 1666, 1612, 1595, 1498, 1358, 1230, 1149and 1022

11c 4-NHSO2Me 404.45 C18H20N4O5S 267e269 (dec.) 3255, 1674, 1610, 1496, 1463, 1328, 1278, 1143and 1089

11d 4-OMe 341.37 C18H19N3O4 243e245 (dec.) 3354, 1654, 1612, 1508, 1483, 1386, 1251, 1095and 1022

N

N

OMeO

MeOX

-n.Bu

(16)

16a 4-NHSO2Me 431.51 C21H25N3O5S 184e189 3401, 2931, 1695, 1633, 1602, 1377, 1272, 1166and 1025

16b 3-NHSO2Me 431.51 C21H25N3O5S 125e127 3419, 2933, 1697, 1639, 1602, 1357, 1272, 1178and 1010

16c 4-NHCOMe 395.46 C22H25N3O4 198e199 3282, 2931, 1668, 1645, 1600, 1533, 1265, 1178and 1033

16f 4-(1H-tetrazol-5-yl) 406.45 C21H22N6O3 220e223 2931, 1670, 1608, 1498, 1438, 1377, 1240, 1078and 1022

16g 3-(1H-tetrazol-5-yl) 406.45 C21H22N6O3 140e145 2933, 1655, 1612, 1498, 1396, 1346, 1253, 1076and 1010

N

NMeO

MeO

N3

X

(18)

18b 2-Cl 341.76 C16H12ClN5O2 190e192 1618, 1606, 1566, 1498, 1436, 1369, 1249, 1174and 1033

18c 3-Cl 341.76 C16H12ClN5O2 205e207 1618, 1606, 1566, 1498, 1436, 1369, 1249, 1174and 1033

18d 3-OMe 337.34 C17H15N5O3 185e187 1618, 1606, 1596, 1498, 1431, 1369, 1253, 1145and 1050

18e 4-OMe 337.34 C17H15N5O3 211e213 1606, 1573, 1434, 1369, 1315, 1184, 1147, 1087and 1026

18f 3-Me 321.34 C17H15N5O2 221e223 1619, 1596, 1502, 1469, 1419, 1378, 1245, 1176and 1030

18g 4-Me 321.34 C17H15N5O2 218e220 1616, 1500, 1463, 1436, 1365, 1278, 1249, 1184and 1029

(continued on next page)

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243 235

Table 1 (continued )

Structures No. X Mol. wt. Mol. formula Mp (�C) IR (KBr)cm�1

N

N

NH2MeO

MeO X

(19)

19b 2-Cl 315.76 C16H14ClN3O2 210e212 (dec.) 3315, 3138, 1654, 1569, 1382, 1284, 1242, 1118and 1028

19c 3-Cl 315.76 C16H14ClN3O2 254e256 (dec.) 3310, 3125, 1635, 1616, 1596, 1310, 1236, 1165and 1022

19d 3-OMe 311.34 C17H17N3O3 240e243 (dec.) 3350, 3228, 1662, 1575, 1508, 1284, 1247, 1107and 1039

19e 4-OMe 311.34 C17H17N3O3 240e241 (dec.) 3477, 3377, 1635, 1552, 1433, 1330, 1236, 1180and 1022

19f 3-Me 295.34 C17H17N3O2 218e220 (dec.) 3388, 3268, 1650, 1558, 1486, 1288, 1234, 1176and 1019

19g 4-Me 295.34 C17H17N3O2 200e202 (dec.) 3477, 3377, 1641, 1558, 1510, 1322, 1257, 1170and 1022

N

N

NH2

MeO

MeO NH

X

(22)

22a 4-NO2 341.33 C16H15N5O4 266e268 (dec.) 3450, 3353, 1676, 1533, 1340, 1278, 1236, 1184and 1029

22b

NHSO2Me465.53 C23H23N5O4S 264e268 (dec.) 3345, 3254, 1658, 1577, 1365, 1270, 1242, 1149

and 1018

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243236

253e255 �C (dec.); IR (KBr): 3336, 1656, 1637, 1614, 1529 1259, 1074and 1030 cm�1.

4.1.4. 4,5-Dimethoxy-2-(3-methoxybenzamido)benzamide (3d)Following a procedure identical to that of 3b, but using 3-anisic

acid (1d) (0.6 g, 3.93 mmol), compound 3dwas obtained as a whitesolid (0.56 g, 48%): mp 207e210 �C (dec.); IR (KBr): 3336, 3193,1658,1633,1614,1533,1483,1278 and 1030 cm�1; 1H NMR: 13.11 (s,1H, NeH), 8.64 (s, 1H, AreH), 8.01 (b, 1H, NeH), 7.61e7.59 (m, 2H,AreH), 7.42e7.38 (m, 2H, AreH), 7.09e7.07 (m, 1H, AreH), 6.52 (b,1H, NeH), 3.98 (s, 3H, OeCH3), 3.93 (s, 3H, OeCH3) and 3.88 (s, 3H,OeCH3).

4.1.5. 4,5-Dimethoxy-2-(4-methoxybenzamido)benzamide (3e)Following a procedure identical to that of 3b but using 4-anisic

acid (1e) (0.6 g, 3.93 mmol), compound 3e was obtained as a paleyellow solid (0.60 g, 51%): mp 215e218 �C (dec.); IR (KBr): 3361,3246, 1670, 1627, 1602, 1525, 1258, 1072 and 1037 cm�1.

4.1.6. 4,5-Dimethoxy-2-(3-methylbenzamido)benzamide (3f)Following a procedure identical to that of 3b but using 3-toluic

acid (1f) (0.53 g, 3.93 mmol), compound 3fwas obtained as a white

Table 2Cytotoxicity data of tested compounds.

Compd % of cells killed at 10 mM Compd % of cells killed at 10 mM

HCT116p53þ/þ

HCT116p53�/�

HEY HCT116p53þ/þ

HCT116p53�/�

HEY

4a 16 22 12 18b 50 63 434c 35 37 42 18c 56 45 354e 10 3 2 18d 25 24 137a 59 50 35 18e 58 48 427b 63 57 49 18f 55 71 427c 80 79 66 18g 47 66 2911a 30 8 0.5 19b 18 e 411b 34 31 23 19c 19 2 311c 28 14 9 19d 19 20 711d 38 22 19 19e 21 42 1716a 6 4 0.5 19f 20 27 616b 26 27 9 19g 5 16 216c 19 17 6 22a 42 22 416f 15 8 2 22b 95 95 9416g 13 3 4

solid (0.48 g, 43%): mp 230e235 �C (dec.); IR (KBr): 3417, 3336,1656, 1635, 1616, 1533, 1377, 1261 and 1041 cm�1.

4.1.7. 4,5-Dimethoxy-2-(4-methylbenzamido)benzamide (3g)Following a procedure identical to that of 3b, but using 4-toluic

acid (1g) (0.53 g, 3.93 mmol), compound 3g was obtained as a paleyellow solid (0.63 g, 56%): mp 246e248 �C (dec.); IR (KBr): 3452,3365, 1666, 1639, 1479, 1386, 1267, 1086 and 1030 cm�1; 1H NMR:13.22 (s, 1H, NeH), 8.60 (s, 1H, AreH), 8.20 (b, 1H, NeH), 7.90e7.88(d, 2H, AreH; J¼ 8.2 Hz), 7.44 (s, 1H, AreH), 7.31e7.29 (d, 2H, AreH;J¼ 8.0 Hz), 7.25 (b, 1H, NeH), 3.93 (s, 3H, OeCH3), 3.89 (s, 3H,OeCH3) and 2.43 (s, 3H, CH3).

4.1.8. N-[3-(3,4-Dihydro-6,7-dimethoxy-4-oxoquinazolin-2-yl)phenyl]acetamide (4a)

Compound 3a (0.5 g, 1.47 mmol) was taken in an empty ther-mometer pocket and fused in a boiling digol bath for 1 h. The solidobtained upon cooling the reaction mixture was triturated withethyl acetate (10 mL) and filtered. The residue thus obtained waspassed through a purifying column of silica (100e190 mesh) using2% methanol in chloroform as eluent. Compound 4a was obtainedas a white solid (0.14 g, 30%): FAB-MS: m/z¼ 340.1 (MþHþ). Anal.Calcd. for C18H17N3O4: C, 63.71; H, 5.05; N, 12.38. Found C, 63.92; H,5.31; N, 12.52%.

Table 3Cytotoxic concentration of select compounds in HCT116 p53þ/þ and HCT116p53�/�colon cancer cell lines.

Compd IC50 (mM) Compd IC50 (mM)

HCT116p53þ/þ

HCT116p53�/�

HCT116p53þ/þ

HCT116p53�/�

7a 9� 0.6 >10 18e z10 7� 37b 9� 1 >10 18f 9� 1 >107c 1.7� 1 0.7� 0.2 18g 8� 0.3 9� 0.718b 8� 0.8 >10 22b 6� 0.05 5� 0.318c z10 z10

Cytotoxic concentration (IC50) is defined as drug concentration causing a 50%decrease in cell population using MTT assay as described in the Experimentalsection.

Fig. 2. Treatment of colon cancer cell lines with compounds 7c and 22b resulted in a significant inhibition of colony formation. At doses above 5 and 1 mM respectively, compounds7c and 22b abolished cell growth.

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243 237

4.1.9. 6,7-Dimethoxy-2-(2-chlorophenyl)-quinazolin-4(3H)-one(4b)

Amixture of diamide 3b (0.8 g, 2.38 mmol), methanol (5 mL) andaq sodium hydroxide solution (50%, 0.5 mL) was refluxed for 2 h.Excess of methanol was recovered and the residue was quenched incold water (10 mL), and neutralized with dilute hydrochloric acid(5%). The precipitate formed was filtered, washed with water anddried. The residue thus obtained was recrystallized from chloroformmethanol mixture to afford compound 4b as a solid (0.5 g, 58%): 1HNMR: d 7.55e7.05 (m, 7H, AreH and NeH), 3.97 (s, 6H, OeCH3); FAB-MS:m/z¼ 317.2 (MþHþ). Anal. Calcd. for C16H13ClN2O3: C, 60.67; H,4.14; N, 8.84. Found C, 60.80; H, 4.30; N, 9.15%.

4.1.10. 2-(3-Chlorophenyl)-6,7-dimethoxyquinazolin-4(3H)-one(4c)

Following a procedure identical to the one as described for 4a,but using diamide 3c (0.50 g, 1.52 mmol), compound 4c was ob-tained as a pale yellow solid (0.19 g, 43%): 1H NMR: d 12.41 (s, 1H,NeH), 8.25 (m, 1H, AreH), 8.15 (m, 1H, AreH), 7.56 (s, 1H, AreH),7.50 (m, 2H, AreH), 7.22 (s, 1H, AreH), 4.00 (s, 3H, OeCH3) and3.97 (s, 3H, OeCH3); FAB-MS:m/z¼ (MþHþ): 317.8. Anal. Calcd. forC16H13ClN2O3: C, 60.67; H, 4.14; N, 8.84. Found C, 60.32; H, 4.50; N,9.04%.

4.1.11. 6,7-Dimethoxy-2-(3-methoxyphenyl)quinazolin-4(3H)-one(4d)

Following a procedure identical to that of 4b, but using diamide3d (0.50 g, 1.51 mmol), compound 4dwas obtained as a pale yellow

solid (0.3 g, 64%): 1H NMR: 12.22 (s, 1H, NeH), 7.79e7.76 (m, 2H,AreH), 7.58 (s, 1H, AreH), 7.42e7.38 (m, 1H, AreH), 7.21 (s, 1H,AreH), 7.06e7.04 (m, 1H, AreH), 4.04 (s, 3H, OeCH3), 4.01 (s, 3H,OeCH3) and 3.91 (s, 3H, OeCH3); FAB-MS: m/z¼ 312 (Mþ).

4.1.12. 6,7-Dimethoxy-2-(4-methoxyphenyl)quinazolin-4(3H)-one(4e)

A mixture of diamide 3e (0.5 g, 1.51 mmol) and freshly preparedsodium t-butoxide (0.43 g, 4.53 mmol) in ethylene glycol (1 mL)was exposed to microwave irradiations (300 W, 190 �C) for 1 min.The cooled reaction mixture was quenched in cold water (30 mL)and neutralized with dilute hydrochloric acid (10%). The precipitateso obtained was filtered, washed with cold water and dried to givecompound 4e as a white solid (0.43 g, 90%): 1H NMR: 12.90 (s, 1H,NeH), 8.64 (s, 1H, AreH), 8.01e7.99 (m, 2H, AreH), 7.34 (s, 1H,AreH), 7.00e6.97 (m, 2H, AreH), 3.99 (s, 3H, OeCH3), 3.93 (s, 3H,OeCH3) and 3.88 (s, 3H, OeCH3); FAB-MS: m/z¼ 312.1 (Mþ). Anal.Calcd. for C17H16N2O4: C, 65.38; H, 5.16; N, 8.97. Found C, 65.70; H,5.20; N, 8.82%.

4.1.13. 6,7-Dimethoxy-2-m-tolylquinazolin-4(3H)-one (4f)Following a procedure identical to that of 4b, but using diamide

3f (0.7 g, 2.23 mmol), compound 4f was obtained as a pale yellowsolid (0.5 g, 77.0%): 1H NMR: 12.18 (s, 1H, NeH), 7.75 (m, 2H, AreH),7.44 (s, 1H, AreH), 7.40 (m, 1H, AreH), 7.09 (s, 1H, AreH), 6.96 (m,1H, AreH) 4.02 (s, 3H, OeCH3), 3.90 (s, 3H, OeCH3) and 2.20 (s, 3H,CH3); FAB-MS:m/z¼ 297.3 (MþHþ). Anal. Calcd. for C17H16N2O3: C,68.91; H, 5.44; N, 9.45. Found C, 68.70; H, 5.20; N, 9.40%.

Fig. 3. Flow cytometric analysis of the cell cycle profiles of HCT116 p53�/�cells treated with compound 22b. HCT116 p53�/�cells were treated for 24 and 48 h with 1 mM of 22b. Cellcycle arrest in G2/M phase was observed. Control cells shown were measured at 24 h and no significant changes were observed.

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243238

4.1.14. 6,7-Dimethoxy-2-p-tolylquinazolin-4(3H)-one (4g)Following a procedure identical to that of 4b, but using diamide

3g (0.5 g, 1.59 mmol), compound 4g was obtained as a pale yellowsolid (0.3 g, 64%): 1H NMR: 12.09 (s, 1H, NeH), 8.09e8.07 (d, 2H,AreH; J¼ 8.3 Hz), 7.58 (s, 1H, AreH), 7.32e7.30 (d, 2H, AreH;J¼ 8.0 Hz), 7.19 (s, 1H, AreH), 4.01 (s, 3H, OeCH3), 3.98 (s, 3H,OeCH3) and 2.43 (s, 3H, CH3); FAB-MS: m/z¼ 297.1 (MþHþ). Anal.Calcd. for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45. Found C, 68.81; H,5.56; N, 9.62%.

4.1.15. 2-[2-(2-Chlorophenyl)acetamido]-4,5-dimethoxybenzamide(6a)

Following a procedure identical to the one as described for 3b,but using compound 2 (0.7 g, 3.57 mmol) and 2-chlorophenylaceticacid (5a) (0.7 g, 3.93 mmol) as starting materials, compound 6awasobtained as a white solid (0.2 g, 16%): mp 190e195 �C (dec.); IR(KBr): 3394, 3276, 1679, 1654, 1618, 1525, 1386, 1271 and1040 cm�1; 1H NMR: 12.19 (s, 1H, NeH), 8.39 (s, 1H, AreH), 7.96 (b,1H, NeH), 7.42e7.38 (m, 2H, AreH), 7.29e7.24 (m, 3H, AreH), 6.75

(b, 1H, NeH), 3.88 (s, 3H, OeCH3), 3.87 (s, 3H, OeCH3) and 3.86 (s,2H, CH2); FAB-MS: m/z¼ 349.8 (MþHþ).

4.1.16. 2-[2-(4-Chlorophenyl)acetamido]-4,5-dimethoxybenzamide(6b)

Following a procedure identical to that of 3b, but usingcompound 2 (0.7 g, 3.57 mmol) and 4-chlorophenylacetic acid (5b)(0.66 g, 3.57 mmol) as starting materials, compound 6b was ob-tained as a white solid (0.3 g, 19%): mp 228e230 �C (dec.); IR (KBr):3382, 3311, 1685, 1645, 1614, 1521, 1265, 1087 and 1039 cm�1; 1HNMR: 12.31 (s, 1H, NeH), 8.34 (s, 1H, AreH), 8.04 (b, 1H, NeH),7.88e7.84 (m, 3H, AreH), 7.35e7.31 (m, 2H, AreH), 7.10 (b, 1H,NeH), 3.86 (s, 3H, OeCH3), 3.85 (s, 3H, OeCH3) and 3.67 (s, 2H,CH2); FAB-MS: m/z¼ 349.7 (MþHþ).

4.1.17. 2-[2-(Naphthalen-1-yl)acetamido]-4,5-dimethoxybenzamide(6c)

Following a procedure identical to that of 3b, but usingcompound 2 (0.7 g, 3.57 mmol) and 1-naphthylacetic acid (5c)

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243 239

(1.00 g, 3.57 mmol) as starting materials, compound 6c was ob-tained as a white solid (1.3 g, 70%): mp 183e185 �C (dec.); IR (KBr):3470, 3224, 1660, 1619, 1525, 1386, 1271, 1085 and 1037 cm�1; 1HNMR: 11.38 (s, 1H, NeH), 8.41 (s, 1H, AreH), 8.08e8.01 (m, 1H,AreH), 7.88e7.83 (m, 2H, AreH), 7.55e7.45 (m, 4H, AreH), 6.83 (s,1H, AreH), 5.62 (s, 2H, NeH), 4.19 (s, 2H, CH2), 3.92 (s, 3H, OeCH3)and 3.68 (s, 3H, OeCH3); FAB-MS: m/z¼ 365.4 (MþHþ).

4.1.18. 2-(2-Chlorobenzyl)-6,7-dimethoxyquinazolin-4(3H)-one(7a)

Following a procedure identical to the one as described for 4e,but using compound 6a (0.2 g, 0.57 mmol) as starting material,compound 7a was obtained as a solid (0.18 g, 95%): 1H NMR: 11.50(s, 1H, NeH), 7.56 (s, 1H, AreH), 7.41e7.39 (m, 1H, AreH), 7.31e7.29(m, 1H, AreH), 7.26e7.23 (m, 2H, AreH), 7.06 (s, 1H, AreH), 4.15 (s,2H, CH2), 4.00 (s, 3H, OeCH3) and 3.96 (s, 3H, OeCH3); FAB-MS: m/z¼ 331.8 (MþHþ). Anal. Calcd. for C17H15ClN2O3: C, 61.73; H, 4.57;N, 8.47. Found C, 61.62; H, 4.71; N, 8.28%.

4.1.19. 2-(4-Chlorobenzyl)-6,7-dimethoxyquinazolin-4(3H)-one(7b)

Following a procedure identical to that of 4e, but usingcompound 6b (0.2 g, 0.57 mmol) as startingmaterial, compound 7bwas obtained as a solid (0.17 g, 89%): 1H NMR: 11.99 (s, 1H, NeH),7.52 (s, 1H, AreH), 7.41e7.37 (m, 2H, AreH), 7.29e7.26 (m, 2H,AreH), 7.09 (s, 1H, AreH), 3.98 (s, 3H, OeCH3), 3.96 (s, 3H, OeCH3)and 3.94 (s, 2H, CH2); FAB-MS:m/z¼ 331.8 (MþHþ). Anal. Calcd forC17H15ClN2O3: C, 61.73; H, 4.57; N, 8.47. Found C, 61.80; H, 4.76; N,8.60%.

4.1.20. 6,7-Dimethoxy-2-[(naphthalen-1-yl)methyl]quinazolin-4(3H)-one (7c)

Following a procedure identical to that of 4e, but usingcompound 6c (0.2 g, 0.55 mmol) as starting material, compound 7cwas obtained as a solid (0.17 g, 93%): 1H NMR: 11.57 (s, 1H, NeH),8.23e8.21 (m, 1H, AreH), 7.88e7.86 (d, 1H, AreH; J¼ 8.1 Hz),7.81e7.79 (d,1H, AreH; J¼ 8.0 Hz), 7.56e7.42 (m, 5H, AreH), 7.08 (s,1H, AreH), 4.47 (s, 2H, CH2), 3.97 (s, 3H, OeCH3) and 3.95 (s, 3H,OeCH3); FAB-MS: m/z¼ 347.4 (MþHþ). Anal. Calcd. forC21H18N2O3: C, 72.82; H, 5.24; N, 8.09. Found C, 72.70; H, 5.34; N,8.26%.

4.1.21. 2-Chloromethyl-6,7-dimethoxyquinazolin-4(3H)-one (9)Chloroacetonitrile (1.17 mL, 18.96 mmol) was added to a satu-

rated solution of dry HCl in dioxane (10 mL). The mixture wasstirred at 0e5 �C for 10 min under anhydrous conditions.Compound 8 (2.0 g, 9.48 mmol) was added into the above solutionand the reaction mixture was stirred at rt for another 24 h. Themixture was quenched in ice-cold water (150 mL) and neutralizedwith aq ammonia. The precipitate formed was filtered, washedwith cold water and dried to afford compound 9 as a pale yellowsolid (1.8 g, 75%): mp 219e223 �C; IR (KBr): 2931, 1670, 1608, 1498,1438, 1377, 1240, 1078 and 1022 cm�1; FAB-MS: m/z¼ 255.7(MþHþ).

4.1.22. 4-[(3,4-Dihydro-6,7-dimethoxy-4-oxoquinazolin-2-yl)methylamino]benzoic acid (11a)

4-Aminobenzoic acid (10a) (0.80 g, 5.89 mmol) and potassiumcarbonate (0.62 g, 5.89 mmol) were added to a solution ofcompound 9 (0.5 g, 1.96 mmol) in DMF (1 mL). The reactionmixture was stirred at rt for 8 h, quenched into cold water andneutralized (pH 4.5e5.0) with dilute hydrochloric acid. Theprecipitate formed was filtered, washed with cold water and dried.The solid thus obtained was recrystallized from a mixture ofchloroform and methanol to afford compound 11a (0.24 g, 35%): 1H

NMR: 12.35 (b, 1H, NeH), 7.71e7.69 (d, 2H, AreH; J¼ 8.7 Hz), 7.43(s, 1H, AreH), 7.09 (s, 1H, AreH), 6.58e6.56 (d, 2H, AreH;J¼ 8.7 Hz), 6.06 (b, 1H, NeH), 5.06 (s, 2H, CH2), 3.87 (s, 3H,OeCH3) and 3.86 (s, 3H, OeCH3); FAB-MS: m/z¼ 356.3 (MþHþ).Anal. Calcd. for C18H17N3O5: C, 60.84; H, 4.82; N, 11.82. Found C,60.60; H, 4.72; N, 11.96%.

4.1.23. 2-[(3-Methanesulfonylamidophenylamino)methyl]-6,7-dimethoxyquinazolin-4(3H)-one (11b)

Following a procedure identical to the one as described for 11a,but using 3-methanesulfonylamidoaniline (10b) (1.09 g,5.89 mmol), compound 11b was obtained as a solid (0.43 g, 54%):1H NMR: 12.26 (b, 1H, NeH), 7.40 (s, 1H, AreH), 7.02 (m, 2H, AreH),5.80 (m, 2H, AreH), 5.59 (m, 1H, AreH), 5.06 (b, 1H, NeH), 4.00 (s,1H, NeH), 3.85 (s, 3H, OeCH3) and 3.80 (s, 3H, OeCH3), 3.44 (m, 2H,CH2), 2.82 (s, 3H, SO2eCH3); FAB-MS: m/z¼ 405.4 (MþHþ). Anal.Calcd. for C18H20N4O5S: C, 53.46; H, 4.98; N, 13.85. Found C, 53.42;H, 5.01; N, 13.80%.

4.1.24. 2-[(4-Methanesulfonylamidophenylamino)methyl]-6,7-dimethoxyquinazolin-4(3H)-one (11c)

Following a procedure identical to that of 11a, but using 4-methanesulfonylamidoaniline (10c) (1.09 g, 5.89 mmol),compound 11c was obtained as a solid (0.5 g, 63%): 1H NMR: 12.30(b, 1H, NeH), 7.40 (s, 1H, AreH), 7.00 (s, 1H, AreH), 6.85 (d, 2H,AreH; J¼ 9.0 Hz), 6.33 (d, 2H, AreH; J¼ 9.0 Hz), 6.06 (b, 1H, NeH),5.06 (s, 2H, CH2), 3.87 (s, 3H, OeCH3) and 3.86 (s, 3H, OeCH3), 2.77(s, 3H, SO2eCH3); FAB-MS: m/z¼ 405.4 (MþHþ). Anal. Calcd. forC18H20N4O5S: C, 53.46; H, 4.98; N, 13.85. Found C, 53.50; H, 5.06; N,13.92%.

4.1.25. 2-[(4-Methoxyphenylamino)methyl]-6,7-dimethoxyquina-zolin-4(3H)-one (11d)

Following a procedure identical to that of 11a, but using 4-anisidine (10d) (0.72 g, 5.89 mmol), compound 11d was obtainedas a solid (0.42 g, 64%): 1H NMR: 12.30 (b, 1H, NeH), 7.48 (s, 1H,AreH), 7.45e7.42 (m, 2H, AreH), 7.19 (s, 1H, AreH), 7.14e7.00 (m,2H, AreH), 6.26 (b, 1H, NeH), 4.82 (s, 2H, CH2), 3.97 (s, 3H, OeCH3),3.88 (s, 3H, OeCH3) and 3.87 (s, 3H, OeCH3); FAB-MS: m/z¼ 342.4(MþHþ). Anal. Calcd. for C18H19N3O4: C, 63.33; H, 5.61; N, 12.31.Found C, 63.56; H, 5.76; N, 12.57%.

4.1.26. N-n-Butyl-4,5-dimethoxy-2-nitrobenzamide (13)A mixture of 4,5-dimethoxy-2-nitrobenzoic acid (12) (5.0 g,

22.0 mmol) and thionyl chloride (15 mL) was refluxed underanhydrous conditions for 2 h. Excess of thionyl chloride wasrecovered under reduced pressure and the residue was dissolved inanhydrous THF (10 mL). This was then added to a solution of n-butylamine (2.6 mL, 26.4 mmol) and triethylamine (TEA, 9.2 mL,66.0 mmol) in THF (15 mL) at 0e5 �C. The reaction mixture wasstirred for 2 h at rt and quenched in cold water. The precipitate soformed was filtered, washed with cold water and dried. The solidthus obtained was crystallized from methanol to obtain yellowcrystals (4.8 g, 77%): mp 130e132 �C; IR (KBr): 3269, 1639, 1519,1340, 1278, 1224, 1180 and 1026 cm�1.

4.1.27. 2-Amino-N-n-butyl-4,5-dimethoxybenzamide (14)A solution of 2-nitro-N-n-butyl-4,5-dimethoxybenzamide (13)

(1.0 g, 3.5 mmol) in methanol (20 mL) was refluxed on a waterbath. Iron powder (1.6 g, 28.4 mmol) and a solution of ammo-nium chloride (1.5 g, 28.3 mmol) in water (2 mL) were addedportion-wise (in 4 parts at an interval of 45 min) to the aboverefluxing solution. Refluxing was continued for 7e8 h and thesolution was filtered through filtering aid and washed with hotmethanol (2�10 mL). The filtrate was concentrated under

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243240

reduced pressure to remove excess of methanol and the resultingaqueous solution was diluted with water (25 mL), basified withsodium bicarbonate (10% aq solution) and extracted with chlo-roform (3� 50 mL). The combined chloroform layer was driedand concentrated to get brown colored residue which was driedunder vacuum to afford compound 14 (0.84 g, 95%): mp109e111 �C; IR (KBr): 3409, 3317, 1635, 1508, 1463, 1373, 1261,1180 and 1026 cm�1.

4.1.28. 4,5-Dimethoxy-N-n-butyl-2-{[4-((methylsulfonyl)amino)phenyl]carboxamido}benzamide (15a)

Following a procedure identical to the one as described for3b, but using compound 14 (0.5 g, 1.98 mmol) and 4-methane-sulfonamidobenzoic acid (1a) (0.47 g, 2.18 mmol), compound 15awas obtained as a pale yellow solid (0.49 g, 55%) and used in thenext step without further purification: mp 184e187 �C; IR (KBr):3460, 1656, 1633, 1614, 1445, 1338, 1245, 1155 and 1010 cm�1.

4.1.29. 4,5-Dimethoxy-N-n-butyl-2-{[3-((methylsulfonyl)amino)phenyl]carboxamido}benzamide (15b)

Following a procedure identical to that of 3b, but using compound14 (0.5 g, 1.98 mmol) and 3-methanesulfonamidobenzoic acid (1b)(0.47 g, 2.18 mmol), compound 15b was obtained as a pale yellowsolid (0.47 g, 54%) and used in the next step without further purifi-cation: mp 221e223 �C; IR (KBr): 3390, 1663, 1640, 1611, 1486, 1332,1257, 1149 and 1038 cm�1.

4.1.30. 2-{[(4-(Acetamido)phenyl)carbonyl]amino}-4,5-Dimethoxy-N-n-butylbenzamide (15c)

Following a procedure identical to that of 3a, but usingcompound 14 (0.5 g, 1.98 mmol) and 4-acetamidobenzoic acid (1c)(0.39 g, 2.18 mmol), compound 15c was obtained as a white solid(0.47 g, 58%) and used in the next step without further purification:mp 115e117 �C; IR (KBr): 3272, 1658, 1643, 1539, 1424, 1322, 1242,1166 and 1019 cm�1.

4.1.31. 2-[4-(Cyanophenyl)carboxamido]-4,5-dimethoxy-N-n-butylbenzamide (15d)

Following a procedure identical to that of 3b, but usingcompound 14 (0.5 g, 1.98 mmol) and 4-cyanobenzoic acid (1d)(0.32 g, 2.18 mmol), compound 15d was obtained as a white solid(0.52 g, 70%) and used in the next step without further purification:mp 190e193 �C; IR (KBr): 3428, 3330, 2233, 1665, 1647, 1618, 1251,1080 and 1024 cm�1.

4.1.32. 2-[3-(Cyanophenyl)carboxamido]-4,5-dimethoxy-N-n-butylbenzamide (15e)

Following a procedure identical to that of 3b, but usingcompound 14 (0.5 g, 1.98 mmol) and 3-cyanobenzoic acid (1e)(0.32 g, 2.18 mmol), compound 15e was obtained as a white solid(0.49 g, 66%) and used in the next step without further purification:mp 184e186 �C; IR (KBr,): 3430, 3345, 2222, 1660, 1640, 1616, 1247,1076 and 1030 cm�1.

4.1.33. 4,5-Dimethoxy-N-n-butyl-2-{[4-(1H-tetrazol-5-yl)phenyl]carboxamido}benzamide (15f)

Sodium azide (0.3 g, 4.88 mmol) and ammonium chloride(0.4 g, 8.14 mmol) were added to the stirred solution ofcompound 15d (0.5 g, 1.31 mmol) in DMF (1 mL). The reactionmixture was heated at 100 �C with stirring for 6 h. The reactionmixture was quenched into cold water and acidified with dilutehydrochloric acid (5%). The precipitate so formed was filtered,washed with water and dried. Pure compound 15f was so ob-tained as white crystals (0.38 g, 68%): mp 220e223 �C; IR (KBr):3359, 1670, 1635, 1596, 1498, 1348, 1269, 1176 and 1010 cm�1; 1H

NMR: 12.72 (b, 1H, NeH), 8.58 (s, 1H, AreH), 8.14 (d, 2H, AreH),7.80 (d, 2H, AreH), 6.92 (s, 1H, AreH), 6.10 (b, 1H, NeH), 4.01 (s,3H, OeCH3), 3.92 (s, 3H, OeCH3), 3.47 (m, 2H, NeCH2), 1.60 (m,2H, CH2), 1.40 (m, 2H, CH2) and 0.98 (t, 3H, CH3); FAB-MS:m/z¼ 425.4 (MþHþ).

4.1.34. 4,5-Dimethoxy-N-n-butyl-2-{[3-(1H-tetrazol-5-yl)phenyl]carboxamido}benzamide (15g)

Following a procedure identical to the one as described for15f,but using compound 15e (0.50 g, 1.31 mmol), compound 15g wasobtained as a white solid (0.41 g, 74%) and used in the next stepwithout further purification: mp 140e145 �C; IR (KBr): 3334, 1666,1635, 1596, 1488, 1356, 1255, 1178 and 1025 cm�1; 1H NMR: 12.87(s, 1H, NeH), 8.75 (s, 1H, AreH), 8.58 (m, 1H, AreH), 8.33e8.31 (d,1H, AreH; J¼ 7.8 Hz), 8.16e8.14 (d, 1H, AreH; J¼ 7.8 Hz), 7.95 (b,1H, NeH), 7.71e7.67 (m, 1H, AreH), 7.27 (s, 1H, AreH), 3.99 (s, 3H,OeCH3), 3.95 (s, 3H, OeCH3), 3.44e3.39 (m, 2H, CH2), 1.65e1.60 (m,2H, CH2), 1.45e1.40 (m, 2H, CH2) and 0.98e0.95 (t, 3H, CH3); FAB-MS: m/z¼ 425.4 (MþHþ).

4.1.35. N-[4-(6,7-Dimethoxy-3-n-butyl-4-oxo-3,4-dihydroquinazolin-2-yl)phenyl]methanesulfonamide (16a)

Following a procedure identical to that of 4b, but usingcompound 15a (0.5 g, 1.11 mmol), compound 16a was obtained asa white solid (0.37 g, 78%): 1H NMR: 7.75 (s, 1H, AreH), 7.50 (m, 1H,AreH), 7.37 (m, 3H, AreH), 7.17 (m, 1H, AreH), 4.00 (m, 8H, 6H- 2�OeCH3 and 2H- CH2), 3.05 (s, 3H, SO2CH3), 1.25 (m, 4H, 2� CH2) and0.80 (m, 3H, CH3); FAB-MS: m/z¼ 432.5 (MþHþ). Anal. Calcd. forC21H25N3O5S: C, 58.45; H, 5.84; N, 9.74. Found C, 58.01; H, 5.67; N,9.88%.

4.1.36. N-[3-(6,7-Dimethoxy-3-n-butyl-4-oxo-3,4-dihydroquinazolin-2-yl)phenyl]methanesulfonamide(16b)

Following a procedure identical to that of 4b, but usingcompound 15b (0.5 g, 1.11 mmol), compound 16b was obtained asa white solid (0.43 g, 82%): 1H NMR: 7.67 (s, 1H, AreH), 7.54e7.52(m, 1H, AreH), 7.42e7.39 (m, 2H, AreH), 7.18e7.16 (m, 1H, AreH),6.78 (s, 1H, AreH), 4.02e3.99 (m, 8H, 6H- 2� OeCH3 and 2H-CH2), 3.09 (s, 3H, CH3), 1.30e1.18 (m, 4H, CH2) and 0.82e0.78 (m,3H, CH3); FAB-MS: m/z¼ 432.5 (MþHþ). Anal. Calcd. forC21H25N3O5S: C, 58.45; H, 5.84; N, 9.74. Found C, 58.63; H, 6.06; N,10.03%.

4.1.37. N-[4-(6,7-Dimethoxy-3-n-butyl-4-oxo-3,4-dihydroquinazolin-2-yl)phenyl]acetamide (16c)

Following a procedure identical to that of 4e, but usingcompound 15c (0.5 g, 1.21 mmol), compound 16c was obtained asa white solid (0.33 g, 69%): 1H NMR: 7.75 (m, 2H, AreH), 7.60 (m,2H, AreH), 7.25 (m, 1H, AreH), 6.80 (s, 1H, AreH), 4.00 (m, 6H, 2�OeCH3), 3.15 (m, 2H, CH2), 2.35 (s, 3H, CH3), 1.60 (m, 2H, CH2), 1.30(m, 2H, CH2) and 1.05 (m, 3H, CH3); FAB-MS: m/z¼ 396.4. Anal.Calcd. for C22H25N3O4: C, 66.81; H, 6.37; N,10.63. Found C, 63.08; H,6.12; N, 10.22%.

4.1.38. 6,7-Dimethoxy-3-butyl-2-[4-(1H-tetrazol-5-yl)phenyl]quinazoline-4(3H)-one (16f)

Following a procedure identical to that of 4e, but usingcompound 15f (0.5 g, 1.31 mmol), compound 16f was obtained asa white solid (0.38 g, 62%): 1H NMR: 7.88e7.86 (d, 2H, AreH;J¼ 8.1 Hz), 7.71 (s, 1H, AreH), 7.58e7.56 (d, 2H, AreH; J¼ 8.2 Hz),7.44 (s, 1H, AreH), 4.07 (s, 3H, OeCH3), 4.03 (s, 3H, OeCH3),3.99e3.97 (m, 2H, CH2), 1.61e1.57 (m, 2H, CH2), 1.19e1.13 (m, 2H,CH2) and 0.78e0.74 (t, 3H, CH3); FAB-MS: m/z¼ 407.4 (MþHþ).Anal. Calcd. for C21H22N6O3: C, 62.06; H, 5.46; N, 20.68. Found C,62.42; H, 5.21; N, 20.28%.

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243 241

4.1.39. 6,7-Dimethoxy-3-n-butyl-2-[3-(1H-tetrazol-5-yl)phenyl]quinazoline-4(3H)-one (16g)

Following a procedure identical to that of 4e, but usingcompound 15g (0.5 g, 1.31 mmol), compound 16g was obtained asa white solid (0.41 g, 74%): 1H NMR: 7.84 (s, 1H, AreH), 7.45 (m, 3H,AreH), 7.30 (s, 1H, AreH), 6.95 (s, 1H, AreH), 4.00 (s, 3H, OeCH3),3.85 (s, 3H, OeCH3), 3.65 (m, 2H, CH2), 1.40 (m, 2H, CH2), 1.33 (m,2H, CH2) and 0.85 (m, 3H, CH3); FAB-MS: m/z¼ 407.4 (MþHþ).Anal. Calcd. for C21H22N6O3: C, 62.06; H, 5.46; N, 20.68. Found C,62.31; H, 5.38; N, 20.32%.

4.1.40. General procedure for the synthesis of chloro derivatives(17be17g)

The synthesis of 4-chloro-2-(2-chlorophenyl)-6,7-dimethoxyquinazoline (17b) is reported as a representativeexample.

A mixture of compound 4b (0.2 g, 0.63 mmol), phosphorusoxychloride (1 mL) and one drop of DMF was refluxed with stirringon an oil bath for 8 h. The reaction mixture was cooled to rt andquenched into crushed ice. The solid obtained was filtered, washedwith cold water and immediately dissolved in chloroform. Theresulting solution was dried over sodium sulfate and concentratedto afford compound 17b as pale yellow solid (0.19 g, 89%), whichwas used in the subsequent step without further purification: mp188e190 �C; IR (KBr): 1612, 1556, 1502, 1444, 1406, 1352, 1236, 1161and 1031 cm�1.

4.1.40.1. 4-Chloro-2-(3-chlorophenyl)-6,7-dimethoxyquinazoline(17c). (0.18 g, 85%); mp. 167e170 �C; IR (KBr): 1618, 1554, 1502,1454, 1404, 1336, 1238, 1164 and 1033 cm�1.

4.1.40.2. 4-Chloro-6,7-dimethoxy-2-(3-methoxyphenyl)quinazoline(17d). (0.64 g, 90%); mp. 188e190 �C; IR (KBr): 1604, 1560, 1514,1438, 1402, 1353, 1255, 1170 and 1020 cm�1.

4.1.40.3. 4-Chloro-6,7-dimethoxy-2-(4-methoxyphenyl)quinazoline(17e). (0.72 g, 85%); mp. 170e172 �C; IR (KBr): 1614, 1562, 1502,1460, 1404, 1336, 1238, 1161 and 1033 cm�1.

4.1.40.4. 4-Chloro-6,7-dimethoxy-2-(3-methylphenyl)quinazoline(17f). (0.17 g, 80%); mp. 230e232 �C; IR (KBr): 1612, 1560, 1500,1438, 1398, 1353, 1253, 1159 and 1020 cm�1.

4.1.40.5. 4-Chloro-6,7-dimethoxy-2-(4-methylphenyl)quinazoline(17g). (0.18 g, 84%); mp. 188e190 �C; IR (KBr): 1616, 1560, 1498,1417, 1398, 1353, 1234, 1159 and 1018 cm�1.

4.1.41. 4-Azido-2-(2-chlorophenyl)-6,7-dimethoxyquinazoline(18b)

Compound 17b (0.7 g, 2.08 mmol) was dissolved in DMF (1 mL)and added sodium azide (0.41 g, 6.35 mmol). The reaction mixturewas exposed to microwave radiations (300W) at 100 �C for 3 min,cooled to rt and quenched into cold water (25 mL). The precipitateformed was filtered, washed with cold water and dried to affordcompound 18b (0.70 g, 68%): 1H NMR: 8.03 (s, 1H, AreH), 7.75 (m,1H, AreH), 7.62 (m, 3H, AreH), 7.54 (m, 1H, AreH), 4.16 (s, 3H,OeCH3) and 4.09 (s, 3H, OeCH3); FAB-MS: m/z¼ 342.7 (MþHþ).Anal. Calcd. for C16H12ClN5O2: C, 56.23; H, 3.54; N, 20.49. Found C,56.36; H, 3.64; N, 20.60%.

4.1.42. 4-Azido-2-(3-chlorophenyl)-6,7-dimethoxyquinazoline(18c)

Following a procedure identical to the one as described for18b,but using compound 17c (0.7 g, 2.08 mmol), compound 18c wasobtained as a pale yellow solid (0.70 g, 68%): 1H NMR: 8.65 (m, 2H,

AreH), 7.99 (s, 1H, AreH), 7.60 (m, 3H, AreH), 4.15 (s, 3H, OeCH3)and 4.12 (s, 3H, OeCH3); FAB-MS: m/z¼ 342.7 (MþHþ). Anal.Calcd for C16H12ClN5O2: C, 56.23; H, 3.54; N, 20.49. Found C, 56.48;H, 3.79; N, 20.68%.

4.1.43. 4-Azido-6,7-dimethoxy-2-(3-methoxyphenyl)quinazoline(18d)

Following a procedure identical to that of 18b, but usingcompound 17d (0.7 g, 2.12 mmol), compound 18d was obtained asawhite solid (0.60 g, 84%): 1H NMR: 8.24 (m,1H, AreH), 8.18 (m,1H,AreH), 7.99 (s, 1H, AreH), 7.80 (s, 1H, AreH), 7.55 (m, 1H, AreH),7.21 (m, 1H, AreH) 4.14 (s, 3H, OeCH3), 4.11 (s, 3H, OeCH3) and3.97 (s, 3H, OeCH3); FAB-MS:m/z¼ 338.3 (MþHþ). Anal. Calcd. forC17H15N5O3:C, 60.53; H, 4.48; N, 20.76. Found C, 60.18; H, 4.62; N,20.63%.

4.1.44. 4-Azido-6,7-dimethoxy-2-(4-methoxyphenyl)quinazoline(18e)

Following a procedure identical to that of 18b, but usingcompound 17e (0.7 g, 2.12 mmol), compound 18e was obtained asa white solid (0.50 g, 70%): 1H NMR: 8.68 (d, 2H, AreH), 7.96 (s, 1H,AreH), 7.55 (s, 1H, AreH), 7.13 (d, 2H, AreH), 4.12 (s, 3H, OeCH3),4.10 (s, 3H, OeCH3) and 3.95 (s, 3H, OeCH3); FAB-MS: m/z¼ 338.3(MþHþ). Anal. Calcd. for C17H15N5O3: C, 60.53; H, 4.48; N, 20.76.Found C, 60.22; H, 4.64; N, 20.66%.

4.1.45. 4-Azido-6,7-dimethoxy-2-m-tolylquinazoline (18f)Following a procedure identical to that of 18b, but using

compound 17f (0.17 g, 0.54 mmol), compound 18f was obtained asa white solid (0.13 g, 75%): 1H NMR: 8.61 (d, 2H, AreH), 7.92 (s, 1H,AreH), 7.50 (s, 1H, AreH), 7.22 (d, 2H, AreH), 4.08 (s, 3H, OeCH3),4.00 (s, 3H, OeCH3) and 3.85 (s, 3H, OeCH3); FAB-MS: m/z¼ 322.3. Anal. Calcd. for C17H15N5O2: C, 63.54; H, 4.71; N, 21.79.Found C, 63.73; H, 4.87; N, 21.52%.

4.1.46. 4-Azido-6,7-dimethoxy-2-p-tolylquinazoline (18g)Following a procedure identical to that of 18b, but using

compound 17g (0.1 g, 0.32 mmol), compound 18g was obtained asa white solid (0.10 g, 98%): 1H NMR: 8.55 (d, 2H, AreH), 7.98 (s, 1H,AreH), 7.58 (s, 1H, AreH), 7.44 (d, 2H, AreH), 4.13 (s, 3H, OeCH3),4.10 (s, 3H, OeCH3) and 2.51 (s, 3H, AreCH3); FAB-MS: m/z¼ 322.3 (MþHþ). Anal. Calcd. for C17H15N5O2: C, 63.54; H, 4.71; N,21.79. Found C, 63.84; H, 4.87; N, 21.66%.

4.1.47. 2-(2-Chlorphenyl)-6,7-dimethoxyquinazolin-4-amine (19b)Compound 18b (0.12 g, 0.35 mmol) was dissolved in methanol

(19 mL) in a three-neck Rb flask (100 mL) equipped witha hydrogen balloon. Palladiumecharcoal (0.1 g, 10%) was added tothe above solution and the reaction mixture was stirred under theatmosphere of hydrogen gas for 4 h. The reaction mixture wasfiltered through filtering aid, washed with hot methanol and thefiltrate was concentrated under reduced pressure to affordcompound 19b as a pale yellow solid (0.10 g, 90%): 1H NMR: 9.4 (b,1H, NeH), 8.52 (m, 1H, AreH), 8.20 (b, 2H, NeH and AreH), 7.96 (s,1H, AreH), 7.58 (m, 3H, AreH), 4.09 (s, 3H, OeCH3) and 4.06 (s, 3H,OeCH3); FAB-MS: m/z¼ 316.7 (MþHþ). Anal. Calcd. forC16H14ClN3O2: C, 60.86; H, 4.47; N, 13.31. Found C, 60.69; H, 4.71; N,13.63%.

4.1.48. 2-(3-Chlorphenyl)-6,7-dimethoxyquinazolin-4-amine (19c)Following a procedure identical to the one as described for 19b,

but using compound 18c (0.60 g, 1.76 mmol) as starting material,compound 19cwas obtained as awhite solid (0.42 g, 76%): 1H NMR:9.04 (b, 2H, NeH2), 8.54 (d, 2H, AreH), 8.30 (b, 1H, AreH), 7.59 (m,3H, AreH), 4.09 (s, 3H, OeCH3) and 4.06 (s, 3H, OeCH3); FAB-MS:

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243242

m/z¼ 316.7 (MþHþ). Anal. Calcd. for C16H14ClN3O2: C, 60.86; H,4.47; N, 13.31. Found C, 60.77; H, 4.12; N, 12.98%.

4.1.49. 6,7-Dimethoxy-2-(3-methoxyphenyl)quinazolin-4-amine(19d)

Following a procedure identical to that of 19b, but usingcompound 18d (0.8 g, 2.37 mmol) as starting material, compound19dwas obtained as awhite solid (0.63 g, 86%): 1H NMR: 8.06e8.02(m, 2H, AreH), 7.48 (s, 1H, AreH), 7.39e7.35 (m, 1H, AreH), 7.32 (s,1H, AreH), 6.99e6.97 (m,1H, AreH), 6.66 (b, 2H, NeH2), 4.04 (s, 3H,OeCH3), 4.02 (s, 3H, OeCH3) and 3.92 (s, 3H, OeCH3); FAB-MS: m/z¼ 312.3 (MþHþ). Anal. Calcd. for C17H17N3O3: C, 65.58; H, 5.50; N,13.50. Found C, 65.73; H, 5.58; N, 13.60%.

4.1.50. 6,7-Dimethoxy-2-(4-methoxyphenyl)quinazolin-4-amine(19e)

Following a procedure identical to that of 19b, but usingcompound 18e (0.3 g, 0.89 mmol) as starting material, compound19ewas obtained as awhite solid (0.24 g, 88%): 1H NMR: 8.43e8.40(m, 2H, AreH), 7.29 (s, 1H, AreH), 7.01e6.98 (m, 2H, AreH), 6.91 (s,1H, AreH), 5.34 (b, 2H, NeH2), 4.04 (s, 3H, OeCH3), 4.00 (s, 3H,OeCH3) and 3.88 (s, 3H, OeCH3); FAB-MS: m/z¼ 312.3 (MþHþ).Anal. Calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50. Found C,65.88; H, 5.66; N, 13.67%.

4.1.51. 6,7-Dimethoxy-2-m-tolylquinazolin-4-amine (19f)Following a procedure identical to that of 19b, but using

compound 18f (0.13 g, 0.39 mmol) as starting material, compound19f was obtained as a white solid (0.10 g, 90.0%): 1H NMR: 8.36 (m,2H, AreH), 7.50 (m, 2H, AreH), 7.28 (m, 2H, AreH), 7.00 (b, 2H,NeH2), 4.05 (s, 3H, OeCH3), 4.03 (s, 3H, OeCH3) and 2.18 (s, 3H,AreCH3); FAB-MS: m/z¼ 296.3 (MþHþ). Anal. Calcd. forC17H17N3O2:C, 69.14; H, 5.80; N, 14.23. Found C, 69.35; H, 5.86; N,14.36%.

4.1.52. 6,7-Dimethoxy-2-p-tolylquinazolin-4-amine (19g)Following a procedure identical to that of 19b, but using

compound 18g (0.1 g, 0.31 mmol) as starting material, compound19gwas obtained as a white solid (0.07 g, 76%): 1H NMR: 8.35e8.33(d, 2H, AreH; J¼ 8.2 Hz), 7.31 (s, 1H, AreH), 7.29e7.27 (d, 2H, AreH;J¼ 8.0 Hz), 6.92 (s, 1H, AreH), 5.45 (b, 2H, NeH2), 4.04 (s, 3H,OeCH3), 3.99 (s, 3H, OeCH3) and 2.42 (s, 3H, CH3); FAB-MS: m/z¼ 296.4 (MþHþ). Anal. Calcd for C17H17N3O2: C, 69.14; H, 5.80; N,14.23. Found C, 69.27; H, 5.76; N, 14.46%.

4.1.53. 6,7-Dimethoxy-N2-(4-nitrophenyl)quinazoline-2,4-diamine(22a)

A mixture of 4-nitroaniline (20a) (0.43 g, 3.12 mmol) and 4-amino-2-chloro-6,7-dimethoxyquoinazoline (21) (0.2 g,0.84 mmol) in DMF (3 mL) was stirred at 100 �C for 16 h. Thereaction mixture was cooled to rt and quenched in diethyl ether(30 mL). The solid thus obtained was filtered, washed with diethylether (2�10 mL) followed by methanol (2�10 mL) and dried. Thesolid residue was recrystallized from a mixture of chloroform andmethanol to afford compound 22a (0.15 g, 43%): 1H NMR:8.09e8.00 (m, 5H, AreH), 6.90 (s, 1H, AreH) 4.15 (bs, 3H, NeH2 andNeH) and 3.94e3.90 (m, 6H, 2� OeCH3); FAB-MS: m/z¼ 342.3(MþHþ). Anal. Calcd. for C16H15N5O4:C, 56.30; H, 4.43; N, 20.52.Found C, 56.02; H, 4.63; N, 20.14%.

4.1.54. N-{4-[(4-Amino-6,7-dimethoxyquinazolin-2-yl)amino]biphenyl-2-yl}methanesulfonamide (22b)

Following a procedure identical to the one as described for 22a,but using 4-amino-20-methanesulfonamidobiphenyl (20b) (1.63 g,6.25 mmol), compound 22bwas obtained as solid (0.81 g, 83%): 1H

NMR: 13.27 (b, 1H, NeH), 10.46 (s, 1H, NeH), 7.88e7.86 (d, 2H,AreH; J¼ 8.6 Hz), 7.81 (b, 1H, NeH), 7.69 (s, 1H, AreH), 7.54e7.52(m, 1H, AreH), 7.43e7.41 (d, 2H, AreH; J¼ 8.6 Hz), 7.39e7.34 (m,1H, AreH), 7.30e7.27 (m, 2H, AreH), 6.96 (s, 1H, AreH), 4.01 (s, 3H,OeCH3), 3.97 (s, 3H, OeCH3) and 2.84 (s, 3H, CH3); FAB-MS: m/z¼ 466.5 (MþHþ). Anal. Calcd. for C23H23N5O4S: C, 59.35; H, 4.98;N, 15.04. Found C, 59.03; H, 4.78; N, 15.43%.

4.2. Cytotoxicity

4.2.1. Cell cultureColon cancer cells, HCT116 p53þ/þ and HCT116 p53�/�were

kindly provided by Dr. Bert Vogelstein (Johns Hopkins MedicalInstitutions, Baltimore, MD). The human ovarian carcinoma cell line(HEY) naturally resistant to cisplatin (CDDP) was kindly providedby Dr. Louis Dubeau (USC Norris Cancer Center). Cells were main-tained as monolayer cultures in RPMI 1640. Media were supple-mented with 10% fetal bovine serum (Gemini-Bioproducts,Woodland, CA) and 2 mmol/L L-Glutamine at 37 �C in a humidifiedatmosphere of 5% CO2. To remove the adherent cells from the flaskfor passaging and counting, cells were washed with PBS withoutcalcium or magnesium, incubated with a small volume of 0.25%trypsineEDTA solution (SigmaeAldrich, St. Louis, MO) for5e10 min, and washed with culture medium and centrifuged. Allexperiments were performed using cells in exponential growth.Cells were routinely checked for Mycoplasma contamination usinga PCR-based assay (Stratagene, Cambridge, UK).

4.2.2. DrugsA 10 mM stock solution of all compounds were prepared in

DMSO and stored at �20 �C. Further dilutions were freshly made inPBS or cell-culture media.

4.2.3. Cytotoxicity assaysCytotoxicity was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay as previously described[13]. Briefly, cells were seeded in 96-well microtiter plates andallowed to attach. Cells were subsequently treated with a contin-uous exposure to the corresponding drug for 72 h. A MTT solution(at a final concentration of 0.5 mg/mL) was added to each well andcells were incubated for 4 h at 37 �C. After removal of the medium,DMSO was added and the absorbance was read at 570 nm. Allassays were done in triplicate. Percentage of cell growth inhibitionwas expressed as: (1� A/C)� 100% (A and C were the absorbancevalues from experimental and control cells, respectively). Inhibitoryconcentration 50% (IC50) values were determined for each drugfrom a plot of log (drug concentration) versus percentage of cellgrowth inhibition. Standard deviation was calculated based on theIC50values obtained from at least three independent experiments.

4.2.4. Colony formation assayColony formation assays were also performed to confirm the

activity of these compounds as described [14]. Briefly, cells wereplated in 6-well plates at a density of 100 cells/well and allowed toattach. Next day, serial dilutions of the corresponding compoundswere added for 24 h. After exposure, cells were washed in PBS andcultured in free media until colonies were formed (8e10 days).Cells were subsequently washed, fixed with a glutaraldehyde 1%solution for 30 min and stained with a solution of crystal violet (2%)for 30 min. After staining, cells were thoroughly washed withwater. Colonies were imaged on the VersaDoc Imaging System(BioRad) and counted using the Quantity One software package(BioRad). The data reported represent the mean of a minimum ofthree independent experiments.

M.R. Yadav et al. / European Journal of Medicinal Chemistry 48 (2012) 231e243 243

4.2.5. Cell cycle analysisCell cycle perturbations were analyzed by propidium iodide

DNA staining. Briefly, exponentially growing cells were treatedwith different doses of the compounds for various times. At the endof each treatment time, cells were collected and washed with PBSafter a gentle centrifugation at 1000 rpm for 5 min. Cells werethoroughly resuspended in 0.5 mL of PBS and fixed in 70% ethanolfor at least 2 h at 4 �C. Ethanol-resuspended cells were thencentrifuged at 1000 rpm for 5 min and washed twice in PBS toremove residual ethanol. For cell cycle analysis, the pellets wereresuspended in 1 mL of PBS containing 0.02 mg/mL of propidiumiodide, 0.5 mg/mL of DNase-free RNase A and incubated at 37 �C for30 min. Cell cycle profiles were obtained using BD LSRII flowcytometer (BD Bioscience, San Jose, CA, USA) and data wereanalyzed with BD FACSDiva software package (BD Biosciences, CA,USA).

References

[1] S.C. Kuo, H.Z. Lee, J.P. Juang, Y.T. Lin, T.S. Wu, J.J. Chang, D. Lednicer, K.D. Paull,C.M. Lin, E. Hamel, K.H. Lee, J. Med. Chem. 36 (1993) 1146e1156.

[2] L. Li, H.K. Wang, S.C. Kuo, T.S. Wu, D. Lednicer, C. Lin, E. Hamel, K.H. Lee, J. Med.Chem. 37 (1994) 1126e1135.

[3] L. Li, H.K. Wang, S.C. Kuo, T.S. Wu, A. Mauger, C. Lin, E. Hamel, K.H. Lee, J. Med.Chem. 37 (1994) 3400e3407.

[4] Y. Xia, Z.Y. Yang, P. Xia, K.F. Bastow, Y. Tachinbana, S.C. Kuo, E. Hamel, T. Hackl,K.H. Lee, J. Med. Chem. 41 (1998) 1155e1162.

[5] Q. Shi, K. Chen, L. Li, J.J. Chang, C. Autry, M. Kozuka, T. Konoshima, J.R. Estes,C.M. Lin, E. Hamel, A.T. McPhail, D.R. McPhail, K.H. Lee, J. Nat. Prod. 58 (1995)475e482.

[6] Y. Xia, Z.Y. Yang, M. Hour, S.C. Kuo, P. Xia, K.F. Bastow, Y. Nakanishi,P. Nampoothiri, T. Kackl, E. Hamel, K.H. Lee, Bioorg. Med. Chem. Lett. 9 (2001)1193e1196.

[7] S.T. Al-Rashood, I.A. Aboldahab, M.N. Nagi, L.A. Abouzeid, A.A. Abdel-Aziz,S.G. Abdel-hamide, K.M. Youssef, A.M. Al-Obaid, H.I. El-Subbagh, Bioorg. Med.Chem. 14 (2006) 8608e8621.

[8] K. Matsuno, T. Seishi, T. Makajima, M. Ichimura, N.A. Giese, J.C. Yu, S. Oda,Y. Nomoto, Bioorg. Med. Chem. Lett. 13 (2003) 3001e3004.

[9] Y. Bathini, I. Singh, P.J. Harvey, P.R. Keller, R. Singh, R.G. MIcetich, D.W. Fry,E.M. Dobrusin, P.L. Toogood, Bioorg. Med. Chem. Lett. 15 (2005) 3881e3885.

[10] S.C. Lin, S.C. Chueh, C.J. Hsiao, T.K. Li, T.H. Chen, C.H. Liao, P.C. Lyu, J.H. Guh,Neoplasia 9 (2007) 830e839.

[11] Aldrich research chemical catalogue 2009e2010, 518654, p. 109.[12] M.-J. Hour, L.-J. Huang, S.-C. Kuo, Y. Xia, K. Bastow, Y. Nakanishi, E. Hamel, K.-

H. Lee, J. Med. Chem. 43 (2000) 4479e4487.[13] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Cancer Res.

47 (1987) 936e942.[14] A. Munshi, M. Hobbs, R.E. Meyn, Methods Mol. Med. 110 (2005) 21e28.